The invention relates to therapeutically active esters and thioesters, processes for preparing them, pharmaceutical compositions containing the same, as well as their uses, i.e., treating proliferative cell growth disorders and inhibiting cells characterized by with hyper-proliferative cell growth. A particular use contemplated by the compounds of the invention lies is the in vitro and in vivo inhibition of growth and proliferation of hyper-proliferative tumourgenic cells such as melanoma and/or lymphoma cells.
Anti-Proliferative Agents
There is a need in cancer therapy for therapeutic compounds, which are inhibitors of the proliferation of tumour cells. One compound, which is known to have such activity, is 5-fluorouracil (5-FU).
Anti-Metastatic and Anti-Invasive Agents
Compounds which have the property of inhibiting the action of the metalloproteinase enzymes involved in connective tissue breakdown and remodeling, such as fibroblast collagenase (Type 1), PMN-collagenase, 72 kDa-gelatinase, 92 kDa-gelatinase, stromelysin, stromelysin-2 and PUMP-1 (known as xe2x80x9cmatrix metalloproteinasesxe2x80x9d, and herein referred to as MMPs) have been proposed and are being tested in the clinic for the treatment of solid tumours.
Cancer cells are particularly adept at utilizing MMPs to achieve rapid remodeling of the extracellular matrix, thereby providing space for tumour expansion and permitting metastasis. Accordingly, MMP inhibitors should minimize these processes and thus slow or prevent cancer progression.
A known class of MMP inhibitors having a hydroxamic acid group as the zinc-binding group may be presented by the structural formula (IA) 
in which the groups R1, to R5. are variable in accordance with the specific prior art disclosures of such compounds. Examples of patent publications disclosing MMP inhibitors of formula (IA) are:
It is noteworthy that all of the compounds embraced by formula (1A) generally act extracellulary and are not shown to enter the target cells i.e., tumourgenic cells, in order to perform their respective functions.
International patent application No. PCT/GB97/02398 describes, inter alia, a method for inhibiting growth and proliferation of tumour cells in mammals. The method comprises administering to a mammal in need thereof an inhibiting amount of a compound of general formula (1) or a pharmaceutically acceptable salt hydrate or solvate thereof sufficient to inhibit proliferation the tumour cells: 
wherein
R is hydrogen or (C1-C6)alkyl;
R1 is hydrogen;
(C2-C6)alkyl;
(C2-C6)alkenyl;
phenyl or substituted phenyl;
phenyl (C1-C6)alkyl or substituted phenyl(C1-C6)alkyl;
phenyl (C2-C6)alkenyl or substituted phenyl(C2-C6)alkenyl
heterocyclyl or substituted heterocyclyl;
heterocyclyl(C1-C6)alkyl or substituted heterocyclyl(C1-C6)alkyl;
a group BSOnA- wherein n is 0, 1 or 2 and B is hydrogen or a (C1-C6)alkyl, phenyl, substituted phenyl, heterocyclyl substituted heterocyclyl, (C1-C6)acyl, phenacyl or substituted phenacyl group, and A represents (C1-C6)alkylene;
hydroxy or (C1-C6)alkoxy;
amino, protected amino, acylamino, (C1-C6)alkylamino or di-(C1-C6)alkylamino; mercapto or (C1-C6)alkylthio;
amino(C1-C6)alkyl, (C1-C6)alkylamino(C1-C6)alkyl, di(C1-C6)alkylamino(C1-C6)alkyl, hydroxy(C1-C6)alkyl, mercapto(C1-C6)alkyl or carboxy(C1-C6)alkyl wherein the amino-, hydroxy-, mercapto- or carboxyl-group are optionally protected or the carboxyl- group amidated;
lower alkyl substituted by carbamoyl, mono(lower alkyl)carbamoyl, di(lower alkyl)carbamoyl, di(lower alkyl)amino, or carboxy-lower alkanoylamino; or
a cycloalkyl, cycloalkenyl or non-aromatic heterocyclic ring containing up to 3 heteroatoms, any of which may be (i) substituted by one or more substituents selected from C1-C6 alkyl, C2-C6 alkenyl, halo, cyano (xe2x80x94CN), xe2x80x94CO2H, xe2x80x94CO2R1 CONH2, xe2x80x94CONHR, xe2x80x94CON(R)2, xe2x80x94OH, xe2x80x94OR, oxoxe2x80x94, xe2x80x94SH, xe2x80x94SR, xe2x80x94NHCOR, and xe2x80x94NHCO2R wherein R is C1-C6 alkyl or benzyl and/or (ii) fused to a cycloalkyl or heterocyclic ring;
R2 is a C1-C12 alkyl,
C1-C12 alkenyl,
C1-C12 alkynyl,
phenyl(C1-C6 alkyl)- ,
heteroaryl(C1-C6 alkyl)- ,
phenyl(C1-C6 alkenyl)- ,
heteroaryl(C2-C6 alkenyl)- ,
phenyl(C2-C6)alkynyl- ,
heteroaryl(C2-C6 alkynyl)- ,
cycloalkyl(C1-C6 alkyl)- ,
cycloalkyl(C2-C6 alkenyl)- ,
cycloalkyl(C2-C6 alkynyl)- ,
cycloalkenyl(C1-C6 alkyl)- ,
cycloalkenyl(C2-C6 alkenyl)- ,
cycloalkenyl(C2-C6 alkynyl)- ,
phenyl(C1-C6 alkyl)O(C1-C6 alkyl)-, or
heteroaryl(C1-C6 alkyl)O(C1-C6 alkyl)- group,
any one of which may be optionally substituted by
C1-C6 alkyl,
C1-C6 alkoxy,
halo,
cyano (xe2x80x94CN),
phenyl, or
phenyl substituted by
C1-C6 alkyl,
C1-C6 alkoxy.
halo, or
cyano (xe2x80x94CN);
R3 is the characterizing group of a natural or non-natural a amino acid in which any functional groups may be protected; and
R4 is an ester or thioester group, or a pharmaceutically acceptable salt, hydrate or solvate thereof.
Objects and Summery of the Invention
An object of the invention resides in providing anti-proliferative ester and thioester""s which inhibit proliferation of rapidly dividing cells that generally attend proliferative cell growth disorders. Exemplary of such disorders are lymphoma, leukemia, myeloma, adenocarcinoma, carcinoma, mesothelioma, teratocarcinoma, choriocarcinoma, small cell carcinoma, large cell carcinoma, melanoma, retinoblastoma, fibrosarcoma, leiomyosarcoma or endothelioma cells.
The ester and thioester compounds in question have certain structural similarities to known MMP inhibitors of general formula (IA) above disclosed in the foregoing patent publications. However, instead of the amide group xe2x80x94CONR4R5, of formula (IA), they have an ester or thioester group. Despite the similarity of structure, it has been shown that compounds of the invention, which have little or no MMP inhibitory activity, are nonetheless potent inhibitors of such cell proliferation, implying a novel mechanism is at work. This anti-proliferation property suggests a utility for the compounds of the present invention in the treatment of cancers. That the compounds of the invention i.e., esters and thioesters maybe used to inhibit growth and proliferation of cells with hyper-proliferative cell growth is neither disclosed nor taught by the prior art.
Although the patent publications listed above predominantly disclose MMP inhibiting compounds of formula (IA), i.e., having an amide group xe2x80x94CONR4R5, a few (WO 92/09563, U.S. Pat. No. 5,183,900, U.S. Pat. No. 5,270,326, EP-A-0489577, EP-A-0489579, WO 93/09097, WO 93/24449, WO 94/25434, WO 94/25435, WO 95/04033, WO 95/19965, and WO 95/22966) include within their generic disclosure compounds having a carboxylate ester group in place of the amide group. The carboxylate ester compounds with which this invention is concerned thus represent a selection of a notional subclass from the compounds proposed in the art as MMP inhibitors, for a specific and previously unrecognized pharmaceutical utility.
WO 95/04033 discloses N4-hydroxy-N1-(1-(S)-methoxycarbonyl-2,2-dimethylpropyl)-2 -(R)-(4-chlorophenylpropyl) succinamide as an intermediate for the preparation of the corresponding methylamide MMP inhibitor.
Likewise, Int. J. Pept. Protein Res. (1996), 48(2), 148-155 discloses the compound:
Phxe2x80x94CH2CH(COxe2x80x94llexe2x80x94OtBu)CH2CONHOH 
The above compound is suggested as an intermediate in the preparation of compounds, which are inhibitors of neurotensin-degrading enzymes. However, those two appear to be the only specific known carboxylate ester compounds of the kind with which this invention is concerned. Neither of the above references specifically suggest the compounds of the present invention or their specific use for inhibiting proliferative cell growth disorders, which are normally attended by hyper-proliferative cell growth.
An alternative embodiment of the invention contemplates an in vivo method for inhibiting growth and proliferation of target cells characterized by hyper-proliferative cell growth. The method comprises administering to a subject in need thereof an inhibiting amount of at least one of the compounds of the invention or a pharmaceutically or veterinarily acceptable sale, hydrate or solvate thereof, in combination with a pharmaceutically or veterinarily acceptable carrier or diluent, sufficient to inhibit proliferation of the target cells.
A still further embodiment is directed to an in vitro method of inhibiting growth and proliferation of cells characterized by hyper-proliferative cell growth, comprising contacting the cells with a growth inhibiting amount of at least one compound of the invention or an acceptable salt, hydrate or solvate thereof.
Another embodiment provides a method for preventing or reducing the onset of a proliferative cell growth disorder in a subject, comprising administering to the subject a therapeutically effective amount of an least one compound of the invention or a pharmaceutically or veterinarily acceptable salt, hydrate or solvate thereof, in combination with a pharmaceutically or veterinarily acceptable carrier or diluent, sufficient to prevent or reduce onset of the disorder.
A still further embodiment of the invention is directed to a method of treating a subject suffering from a proliferative cell growth disorder characterized by hyper-proliferative cell growth. The method comprises administering to the subject a therapeutically effective amount of at least one compound of the invention or a pharmaceutically or veterinarily acceptable salt, hydrate or solvate thereof, in combination with a pharmaceutically or veterinarily acceptable carrier or diluent, sufficient to treat the subject.
A still further embodiment is directed to a pharmaceutical composition useful for treating a proliferative cell growth disorder, comprising a therapeutically effective amount of at least one of the compounds of the invention in combination with a pharmaceutically or a veterinarily acceptable carrier or diluent.
Another embodiment of the invention embraces an anti-proliferation composition comprising an effective amount of at least one of the compounds of the invention or a pharmaceutically or veterinarily acceptable salt, hydrate or solvate thereof, in combination with a pharmaceutically or veterinarily acceptable carrier or diluent.
A process for manufacturing the above compounds and various compositions embraced by the invention is also a subject of the invention.
A final embodiment of the invention provides for a method for the ex-vivo treatment of a proliferative cell growth disorder characterized by hyper-proliferative cell growth. The method provides for extracting a biological sample suspected of containing cells exhibiting hyper-proliferative cell growth from a subject suspected of suffering from a proliferative cell growth disorder, contacting ex-vivo the biological sample with an effective amount of at least one of the compounds of the present invention or a pharmaceutically or veterinarily acceptable salt, hydrate or solvate thereof, sufficient to inhibit cell proliferation of the cells in the biological sample, followed by introducing the treated sample into the subject.
The above and other objects, features and advantages of the present invention will become apparent from the description and examples that follow, it being recognized that the examples in no way limit the scope of the invention claimed.
In its broadest aspect, the present invention provides a method for inhibiting proliferation of tumour cells in mammals, comprising administering to the mammal suffering such proliferation an amount of a compound of general formula (IB) or a pharmaceutically acceptable salt hydrate or solvate thereof sufficient to inhibit such proliferation: 
wherein
R is hydrogen or (C1-C6)alkyl;
R1 is hydrogen;
(C1-C6)alkyl;
(C2-C6)alkenyl;
phenyl or substituted phenyl;
phenyl (C1-C6)alkyl or substituted phenyl(C1-C6)alkyl;
phenyl (C2-C6)alkenyl or substituted phenyl(C2-C6)alkenyl heterocyclyl or substituted heterocyclyl;
heterocyclyl(C1-C6)alkyl or substituted heterocyclyl(C1-C6)alkyl;
a group BSOnA- wherein n is 0, 1 or 2 and B is hydrogen or a (C1-C6 ) alkyl, phenyl, substituted phenyl, heterocyclyl substituted heterocyclyl, (C1-C6)acyl, phenacyl or substituted phenacyl group, and A represents (C1-C6)alkylene;
hydroxy or(C1-C6)alkoxy;
amino, protected amino, acylamino, (C1-C6)alkylamino or di-(C1-C6)alkylamino;
mercapto or (C1-C6)alkylthio;
amino(C1-C6)alkyl, (C1-C6)alkylamino(C1-C6)alkyl, di(C1-C6)alkylamino(C1-C6)alkyl, hydroxy(C1-C6)alkyl, mercapto(C1-C6)alkyl or carboxy(C1-C6)alkyl wherein the amino-, hydroxy-, mercapto- or carboxyl-group are optionally protected or the carboxyl- group amidated;
lower alkyl substituted by carbamoyl, mono(lower alkyl)carbamoyl, di(lower alkyl)carbamoyl, di(lower alkyl)amino, or carboxy-lower alkanoylamino; or a cycloalkyl, cycloalkenyl or non-aromatic heterocyclic ring containing up to 3 heteroatoms, any of which may be (i) substituted by one or more substituents selected from C1-C6 alkyl, C2-C6 alkenyl, halo, cyano (xe2x80x94CN), xe2x80x94CO2H, xe2x80x94CO2R, CONH2, xe2x80x94CONHR, xe2x80x94CON(R)2, xe2x80x94OH, xe2x80x94OR, oxoxe2x80x94, xe2x80x94SH, xe2x80x94SR, xe2x80x94NHCOR, and xe2x80x94NHCO2R wherein R is C1-C6 alkyl or benzyl and/or (ii) fused to a cycloalkyl or heterocyclic ring;
R2is a C1-C12alkyl,
C2-C12alkenyl,
C2-C12alkenyl,
phenyl(C1-C6alkyl)-,
heteroaryi(C1-C6alkyl)-,
phenyl(C2-C6alkenyl)-,
heteroaryl(C2-C6alkenyl)-,
phenyl(C2-C6alkynyl)-,
heteroaryl(C2-C6alkynyly,
cycloalkyl(C1-C6alkyl)-,
cycloalkyl(C2-C6alkenyl)-,
cycloalkyl(C2-C6alkynyl)-,
cycloalkenyl(C1-C6alkyl)-,
cycloalkenyl(C2-C6alkenyl)-,
cycloalkenyl(C2-C6alkynyly,
phenyl(C1-C6alkyl)O(C1-C6alkyl)-, or
heteroaryl(C1-C6 alkyl)O(C1-C6alkyl)- group,
any one of which may be optionally substituted by
C1-C6 alkyl,
C1-C6 alkoxy,
halo,
cyano (xe2x80x94CN),
phenyl, or
phenyl substituted by
C1-C6 alkyl,
C1-C6 alkoxy,
or cyano (xe2x80x94CN);
R3 is the characterizing group of a natural or non-natural xcex1 amino acid in which any functional groups may be protected; and
R4 is an ester or thioester group, or a pharmaceutically acceptable salt, hydrate or solvate thereof.
In another broad aspect of the invention, there is provided the use of a compound of formula (I) as defined in the immediately preceding paragraph, in the preparation of a pharmaceutical composition for inhibiting proliferation of tumour cells in mammals.
The present invention also provides novel compounds of general formula (I) above wherein R, R1, R2, R3 and R4 are as defined above with reference to formula (I), and pharmaceutically acceptable salts, hydrates or solvates thereof, PROVIDED THAT:
(i) when R and R1, are hydrogen, R2 is 4-chlorophenylpropyl, and R3 is tert-butyl, then R4 is not a methyl carboxylate ester group; and
(ii) when R and R4, are hydrogen, R2 is phenylmethyl, and R3 is 1-methylprop-1-yl, then R4 is not a tert-butyl carboxylate ester group.
One particular sub-group of the novel esters and thioesters of the invention consists of compounds of formula (I) above, wherein:
R, R1, and R4 are as defined above with reference to formula (1)
R2 is C1-C12 alkyl, C2-C12alkenyl, C2-C12 alkynyl, biphenyl(C1-C6alkyl)-,
phenyl[heteroaryl(C1-C6 alkyl)-, heteroarylphenyl(C1-C6 alkyl)-,
biphenyl (C2-C6alkenyl)-, phenylheteroaryl (C2-C6alkenyl)-, heteroarylphenyl (C2-C6 alkenyl)-,
phenyl(C2-C6 alkynyl)-, heteroaryl(C2-C6alkynyl)-,
biphenyl(C2-C6alkynyl)-, phenylheteroaryl (C2-C6alkynyl)-, heteroarylphenyl (C2-C6 alkynyl)-,
phenyl(C1-C6 alkyl)O(C1-C6 alkyl)-, or heteroaryi (C1-C6 alkyl)O(C1-C6 alkyl)-,
any one of which may be optionally substituted on a ring carbon atom by C1-C6 alkyl, C1-C6 alkoxy, halo, or cyano (xe2x80x94CN); and
R3 is C1-C6alkyl, optionally substituted benzyl, optionally substituted phenyl, optionally substituted heteroaryl; or
the characterizing group of a natural a amino acid, in which any functional group may be protected, any amino group may be acylated and any carboxyl group present may be amidated; or
a heterocyclic (C1-C6)alkyl group, optionally substituted in the heterocyclic ring;
and pharmaceutically acceptable salts, hydrates or solvates thereof.
As used herein the term xe2x80x9c(C1-C6)alkylxe2x80x9d or xe2x80x9clower alkylxe2x80x9d means a straight or branched chain alkyl moiety having from 1 to 6 carbon atoms, including for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl and n-hexyl.
The term (C2-C6) xe2x80x9calkenylxe2x80x9d means a straight or branched chain alkenyl moiety having from 2 to 6 carbon atoms having at least one double bond of either E or Z stereochemistry where applicable. This term would include, for example, vinyl, allyl, 1- and 2-butenyl and 2-methyl-2-propenyl.
The term xe2x80x9cC2-C6 alkynylxe2x80x9d refers to straight chain or branched chain hydrocarbon groups having from two to six carbon atoms and having in addition one triple bond. This term would include for example, ethynyl,- 1 -propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl.
The term xe2x80x9ccycloalkylxe2x80x9d means a saturated alicyclic moiety having from 3-8 carbon atoms and includes, for example, cyclohexyl, cyclooctyl, cycloheptyl, cyclopentyl, cyclobutyl and cyclopropyl.
The term xe2x80x9ccycloalkenylxe2x80x9d means an unsaturated alicyclic moiety having from 4-8 carbon atoms and includes, for example, cyclohexenyl, cyclooctenyll, cycloheptenyl, cyclopentenyl, and cyclobutenyl. In the case of cycloalkenyl rings of from 5-8 carbon atoms, the ring may contain more than one double bond.
The term xe2x80x9carylxe2x80x9d means an unsaturated aromatic carbocyclic group which is monocyclic (eg phenyl) or polycyclic (eg naphthyl).
The unqualified term xe2x80x9cheterocyclylxe2x80x9d or xe2x80x9cheterocyclicxe2x80x9d means (i) a 5-7 membered heterocyclic ring containing one or more heteroatoms selected from S, N and O, and optionally fused to a benzene ring, including for example, pyrrolyl, furyl, thienyl, piperidinyl, imidazolyl, oxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, benzimidazolyl, maleimido, succinimido, phthalimido, 1,2-dimethyl-3, 5-dioxo-1,2, 4-triazolidin-4-yl, 3,4, 4-trimethyl-2, 5-dioxo-1-imidazolidinyl, 2-methyl-3, 5-dioxo-1,2, 4-oxadiazol-4-yl, 3 methyl-2,4, 5-trioxo-1-imidazolidinyl,2, 5-dioxo-3-phenyl-1-imidazolidinyl, 2-oxo-1-pyrrolidinyl, 2, 5-dioxo-1-pyrrolidinyl or 2, 6-dioxopipeddinyl, or (ii) a napththalimido (i.e. 1, 3-dihydro-1, 3-dioxo-2H-benz[f]isoindol-2-yl), 1, 3-dihydro-1-oxo-2H-benz[f]isoindol-2-yl, 1, 3-dihydro-1, 3-dioxo-2H-pyrrolo[3, 4-b]quinolin-2-yl, or 2, 3-dihydro-1, 3-dioxo-1 H-benz[d,e]isoquinolin-2-yl group.
The term xe2x80x9cheteroarylxe2x80x9d means a 5-7 membered substituted or unsubstituted aromatic heterocycle containing one or more heteroatoms. Illustrative of such rings are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, pyrazolyl, isoxazolyl, isothiazolyl, thizolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl and triazinyl.
The term xe2x80x9cesterxe2x80x9d or xe2x80x9cesterified carboxyl groupxe2x80x9d means a group R9O(C=O)xe2x80x94in which R9 is the group characterizing the ester, notionally derived from the alcohol R5,OH.
The term xe2x80x9cthioesterxe2x80x9d means a group R9S(C=O)- or R9S(C=S)- or R9(C=S)-in which R is the group characterizing the thioester, notionally derived from the alcohol R9OH or the thioalcohol R9SH.
Unless otherwise specified in the context in which it occurs, the term xe2x80x9csubstitutedxe2x80x9d as applied to any moiety herein means substituted with up to four substituents, each of which independently may be (C1-C6)alkyl, (C1-C6)alkoxy, hydroxy, mercapto, (C1-C6)alkylthio, amino, halo (including fluoro, chloro, bromo and iodo), nitro, trifluoromethyl, xe2x80x94COOH, xe2x80x94CONH2, xe2x80x94CN, xe2x80x94COORA, xe2x80x94CONHRA or xe2x80x94CONHRARA wherein RA is a (C1-C6) alkyl group or the residue of a natural alpha-amino acid.
The term xe2x80x9cside chain of a natural or non-natural alpha-amino acidxe2x80x9d means the group R1 in a natural or non-natural amino acid of formula NH2xe2x80x94CH(R1)xe2x80x94COOH.
Examples of side chains of natural alpha amino acids include those of alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, histidine, 5 hydroxylysine, 4-hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, a-aminoadipic acid, xcex1-amino-n-butyric acid, 3,4-dihydroxyphenylalanine, homoserine, xcex1methylserine, ornithine, pipecolic acid, and thyroxine.
Natural alpha-amino acids which contain functional substituents, for example amino, carboxyl, hydroxy, mercapto, guanidyl, imidazolyl, or indolyl groups in their characteristic side chains include arginine, lysine, glutamic acid, aspartic acid, tryptophan, histidine, serine, threonine, tyrosine, and cysteine. When R3 in the compounds of the invention is one of those side chains, the functional substituents may optionally be protected.
The term xe2x80x9cprotectedxe2x80x9d when used in relation to a functional substituent in a side chain of a natural alpha-amino acid means a derivative of such a substituent, which is substantially non-functional. For example, carboxyl groups may be esterified (for example as a C1-C6 alkyl ester), amino groups may be converted to amides (for example as a NHCOC1-C6 alkyl amide) or carbamates (for example as an NHC(=O)O C1-C6 alkyl or NHC(=O)OCH2Ph carbamate), hydroxyl groups may be converted to ethers (for example an O C1-C6, alkyl or a O(C1-C6alkyl)phenyl ether) or esters (for example a OC(=O) C1-C6 alkyl ester) and thiol groups may be converted to thioethers (for example a tert-butyl or benzyl thioether) or thioesters (for example a SC(=O) C1-C6 alkyl thioester).
Examples of side chains of non-natural alpha amino acids include those referred to below in the discussion of suitable R3 groups for use in compounds of the present invention.
The term xe2x80x9cproliferative cell growth disorderxe2x80x9d is a disorder that is characterized by hyper-proliferative cell growth in that such target cells exhibit a growth pattern that is hyper-proliferative (rapidly dividing cells).
Salts of the compounds of the invention include physiologically acceptable acid addition salts for example hydrochlorides, hydrobromides, sulphates, methane sulphonates, p-toluenesulphonates, phosphates, acetates, citrates, succinates, lactates, tartrates, fumarates and maleates. Salts may also be formed with bases, for example sodium, potassium, magnesium, and calcium salts.
There are several chiral centers in the compounds according to the invention because of the presence of asymmetric carbon atoms. The presence of several asymmetric carbon atoms gives rise to a number of diastereomers with R or S sterochemistry at each chiral center. In the compounds of the invention, the C atom carrying the hydroxamic acid and R, groups is predominantly in the S configuration, the C atom carrying the R2 group is predominantly in the R configuration, and the C atom carrying the R3 and R4 groups is in either the R or S configuration, with the predominantly S configuration presently preferred.
As previously stated, the compound with which the present invention is concerned are principally distinguished from the compounds disclosed in the prior patent publications listed above by the ester or thioester group R4. Accordingly the groups R, R1, R1, R2, and R3, may include those which have been disclosed in the corresponding positions of compounds disclosed in any of those prior art patent publications listed above. Without limiting the generality of the foregoing, examples of substituents; R, R1, R1, R2, and R3 are given below:
The group R1 
R1 may be, for example,
hydrogen, methyl, ethyl, n-propyl, n-butyl, isobutyl, hydroxyl, methoxy, allyl, phenylpropyl, phenylprop-2-enyl, thienylsulphanylmethyl, thienylsulphinylmethyl, or thienylsulphonylmethyl; or
C1-C4 alkyl, eg. methyl, ethyl n-propyl or n-butyl, substituted by a phthalimido, 1,2-dimethyl-3,5-dioxo-1,2,4-triazolidin-4-yl, 3-methyl-2,5-dioxo-1 imidazolidinyl, 3,4,4-trimethyl-2,5-dioxo-1-imidazolidinyl, 2-methyl-3,5-dioxo-1,2,4-oxadiazol-4-yl, 3-methyl-2,4,5-trioxo-1-imidazolidinyl, 2,5-dioxo-3-phenyl-1-imidazolidinyl, 2-oxo-1-pyrrolidinyl, 2,5-dioxo-1-pyrrolidinyl or 2,6-dioxopiperidinyl, 5,5-dimethyl-2,4-dioxo-3-oxazolidinyl, hexahydro-1,3 dioxopyrazolol[1,2,a][1,2,4]-triazol-2-yl, or a naphththalimido (ie. 1,3-dihydro-1,3-dioxo-2H-benz[f]isoindol-2-yl), 1,3-dihydro-1-oxo-2H-benz[f]lisoindol-2-yl, 1,3-dihydro-1,3-dioxo-2H-pyrrolo[3,4-b]quinolin-2-yl, or 2,3-dihydro-1,3-dioxo 1H-benz[d,e]isoquinolin-2-yl group; or
cyclohexyl, cyclooctyl, cycloheptyl, cyclopentyl, cyclobutyl, cyclopropyl, tetrahydro-pyra nyl or morpholinyl.
Presently preferred R1 groups include n-propyl, allyl, methoxy and thienylsulfanylmethyl.
The group R2 
R2 may for example be
C1-C6alkyl, C3-C6 alkenyl or C3-C6 alkynyl; phenyl(C1-C6 alkyl)-, phenyl(C3-C6 alkenyl)- or phenyl(C3-C6alkynyl)- optionally substituted in the phenyl ring;
heteroaryl(C1-C6alkyl)-, heteroaryl(C3-C6 alkenyl)- or heteroaryl(C1-C6 alkynyl)- optionally substituted in the heteroaryl ring;
4-phenylphenyl(C1-C6 alkyl)-, 4-phenylphenyl(C3-C6alkenyl)-, 4-phenylphenyl(C3-C6alkynyl)-, 4-heteroarylphenyl(C1-C6 alkyl)-, 4-heteroarylphenyl(C3-C6 alkenyl)-, 4-heteroarylphenyl(C3-C6alkynyl)-, optionally substituted in the terminal phenyl or heteroaryl ring;
phenoxy (C1-C6 alkyl)- or heteroaryloxy(C1-C6 alkyl)- optionally substituted in the phenyl or heteroaryl ring;
Specific examples of such groups include methyl, ethyl, n- and iso-propyl, n-, iso and tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-nonyl, n-decyl, prop-2-yn-1-yl, 3-phenylprop-2-yn-1-yl, 3(2-chlorophenyl)prop-2-yn-1-yl, phenylpropyl, 4-chlorophenylpropyl, 4-methylphenylpropyl, 4-methoxyphenylpropyl, phenoxybutyl, 3-(4-pyridylphenyl)propyl-, 3-(4-(4-pyridyl)phenyl)prop-2-yn-1-yl, 3-(4-phenylphenyl)propyl-, 3-(4-phenyl)phenyl)prop-2-yn-1-yl and 3-[(4-chlorophenyl)phenyl]propyl-.
Presently preferred R2 groups include isobutyl, n-hexyl, 3-(2-chlorophenyl)prop-2-yn-1-yl.
The group R3 
R3 may for example be C1-C6 alkyl, phenyl, 2,- 3-, or 4-hydroxyphenyl, 2,- 3-, or 4-methoxyphenyl, 2,- 3-, or 4-pyridylmethyl, benzyl, 2,- 3-, or 4-hydroxy-benzyl, 2,- 3-, or 4-benzyloxybenzyl, 2,- 3-, or 4-C1-C6-alkoxybenzyl, or
benzyloxy(C1-C6 alkyl)-group; or
the characterizing group of a natural xe2x88x9d amino acid, in which any functional group may be protected, any amino group may be acylated and any carboxyl group present may be amidated; or
a group xe2x80x94[Alk]nR6 where Alk is a (C1-C6)alkyl or (C2-C6)alkenyl group optionally interrupted by one or more xe2x80x94Oxe2x80x94, or xe2x80x94Sxe2x80x94 atoms or xe2x80x94N(R7)xe2x80x94 groups [where R7 is a hydrogen atom or a (C1-C6)alkyl group], n is 0 or 1, and R6 is an optionally substituted cycloalkyl or cycloalkenyl group; or
a benzyl group substituted in the phenyl ring by a group of formula xe2x80x94OCH2COR8 where R8 is hydroxyl, amino, (C1-C6)alkoxy, phenyl(C1-C6)alkoxy, (C1-C6)alkylamino, di((C1-C6)alkyl)amino, phenyl(C1-C6)alkylamino, the residue of an amino acid or acid halide, ester or amide derivative thereof, said residue being linked via an amide bond, said amino acid being selected from glycine, xcex1 or xcex2 alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, histine, arginine, glutamic acid, and aspartic acid; or
a heterocyclic(C1-C6)alkyl group, either being substituted or mono- or di substituted in the heterocyclic ring with halo, nitro, carboxy, (C1-C6)alkoxy, cyano, (C1-C6)alkanoyl, trifluoromethyl(C1-C6)alkyl, hydroxy, formyl, amino, (C1-C6)alkylamino, di-(C1-C6)alkylamino, mercapto, (C1-C6)alkylthio, hydroxy(C1-C6)alkyl, mercapto(C1-C6)alkyl or (C1-C6)alkylphenylmethyl; or
a group xe2x80x94CRaRbRc, in which:
each of Ra, Rb and Rc is independently hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyl(C1-C6)alkyl, (C1-C6)cycloalkyl; or
Rc is hydrogen and Ra and Rb are independently phenyl or heteroaryl Such as pyridyl; or
Rc is hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyl(C1-C6)alkyl, or (C3-C8)cycloalkyl, and Ra and Rb together with the carbon atom to which they are attached form a 3 to 8 membered cycloalkyl or a 5- to 6-membered heterocyclic ring; or
Ra, Rb and Rc together with the carbon atom to which they are attached form a tricyclic ring (for example adamantyl); or
Ra and Rb are each independently (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyl(C1-C6)alkyl, or a group as defined for Rc below other than hydrogen, or Ra and Rb together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic ring, and Rc is hydrogen, xe2x80x94OH, xe2x80x94SH, halogen, xe2x80x94CN, xe2x80x94CO2H, (C1-C4)perfluoroalkyl, xe2x80x94CH2OH, xe2x80x94CO2(C1-C6)alkyl, xe2x80x94O(C1-C6)alkyl, xe2x80x94O(C2-C6)alkenyl, xe2x80x94S(C1-C6)alkyl, xe2x80x94SO(C1-C6)alkyl, xe2x80x94SO2(C1-C6)alkyl, xe2x80x94S(C2-C6)alkenyl, xe2x80x94SO(C2-C6)alkenyl, xe2x80x94SO2(C2-C6)alkenyl or a group xe2x80x94Qxe2x80x94W wherein Q represents a bond or xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94 or xe2x80x94SO2xe2x80x94 and W represents a phenyl, phenylalkyl, C3-C8)cycloalkyl, (C3-C8)cycloalkylalkyl, (C4-C8)cycloalkenyl, (C4-C8)cycloalkenylalkyl, heteroaryl or heteroarylalkyl group, which group W may optionally be substituted by one or more substituents independently selected from, hydroxyl, halogen, xe2x80x94CN, xe2x80x94CO2H, xe2x80x94CO2(C1-C6)alkyl, xe2x80x94CONH2, xe2x80x94CONH(C1-C6)alkyl, xe2x80x94CONH(C1-C6alkyl)2, xe2x80x94CHO, xe2x80x94CH2OH, (C1-C4)perfluoroalkyl, xe2x80x94O(C1-C6)alkyl, xe2x80x94S(C1-C6)alkyl, xe2x80x94SO(C1-C6)alkyl, xe2x80x94SO2(C1-C6)alkyl, xe2x80x94NO2, xe2x80x94NH2, xe2x80x94NH(C1-C6)alkyl, xe2x80x94N((C1-C6)alkyl)2, xe2x80x94NHCO(C1-C6)alkyl, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C3-C8)cycloalkyl, (C4-C8)cycloalkenyl, phenyl or benzyl.
Examples of particular R3 groups include benzyl, phenyl, cyclohexylmethyl, pyridin-3-ylmethyl, tert-butoxymethyl, iso-butyl, sec-butyl, tert-butyl, 1-benzylthio-1-methylethyl, 1-methylthio-1-methylethyl, and 1-mercapto-1-methylethyl.
Presently preferred R3 groups include phenyl, benzyl, tert-butoxymethyl and isobutyl.
The group R4 
Examples of particular ester and thioester groups R4 groups include those of formula xe2x80x94(Cxe2x95x90O)OR9, xe2x80x94(Cxe2x95x90O)SR9, xe2x80x94(Cxe2x95x90S)SR9, and xe2x80x94(Cxe2x95x90S)R9 wherein R9 is (C1-C6)alkyl, (C2-C6)alkenyl, cycloalkyl, cycloalkyl(C1-C6)alkyl-, phenyl, heterocyclyl, phenyl(C1-C6)alkyl-, heterocyclyl(C1-C6)alkyl-, (C1-C6)alkoxy(C1-C6)alkyl-, (C1-C6)alkoxy(C1-C6alkoxy(C1-C6)alkyl-, any of which may be substituted on a ring or non-ring carbon atom or on a ring heteroatom, if present. Examples of such R9 groups include methyl, ethyl, n- and iso-propyl, n-, sec- and tert-butyl, 1-ethyl-prop-1-yl, 1-methyl-prop-1-yl, 1-methyl-but-1-yl, cyclopentyl, cyclohexyl, allyl, phenyl, benzyl, 2-, 3- and 4-pyridylmethyl, N-methylpiperidin-4-yl, 1-methylcyclopent-1 yl, adamantyl, tetrahydrofuran-3-yl and methoxyethyl.
Presently preferred are compounds of formula (1B) wherein R4 is a carboxylate ester of formula-(Cxe2x95x90O)OR9, wherein R9 is benzyl, cyclopentyl, isopropyl or tert-butyl.
The group R
Presently preferred R groups are hydrogen and methyl.
Specific examples of compounds of the invention include those prepared according to Examples 1-3 and 5-43 below, and salts, hydrates and solvates thereof.
Compounds presently preferred for their potencies as inhibitors of proliferation of various rapidly dividing tumor cells are:
2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionic acid cyclopentyl ester,
2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionic acid benzyl ester,
2S-{2R-[1S-Hydroxycarbamoyl-2-(thiophen-2-yl sulphanyl))-ethyl]-4-methyl-pentanoylamino}-3-phenyl-propionic acid isopropyl ester,
2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-4-methyl-pentanoic acid cyclopentyl ester, and p0 pharmaceutically acceptable salts, hydrates and esters thereof.
Compounds according to the present invention wherein R4 is a carboxylate ester group may be prepared by a process comprising causing an acid of general formula (II) 
or an activated derivative thereof to react with hydroxylamine, O-protected hydroxylamine, or an N,O-diprotected hydroxylamine, or a salt thereof, R, R1, R2, R3, and R4 being as defined in general formula (I) except that any substituents in R1, R2, R3, and R4 which are potentially reactive with hydroxylamine, O-protected hydroxylamine, the N,O-diprotected hydroxylamine or their salts may themselves be protected from such reaction, then removing any protecting groups from the resultant hydroxamine acid moiety and from any protected substituents in R1, R2, R3 and R4.
Conversion of (II) to an activated derivative such as the pentafluorophenyl, hydroxycussinyl, or hydroxybenzotriazolyl ester may be effected by reaction with the appropriate alcohol in the presence of a dehydrating agent such as dicyclohexyl dicarbodiimide (DCC), N,N-dimethylaminopropyl-Nxe2x80x2-ethyl carbodiimide (EDC), or 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ).
Protecting groups as referred to above are well known per se, for example from the techniques of peptide chemistry. Amino groups are often protectable by benzyloxycarbonyl, t-butoxycarbonyl or acetyl groups, or in the form of a phthalimido group. Hydroxy groups are often protectable as readily cleavable ethers such as the t-butyl or benzyl ether, or as readily cleavable esters such as the acetate. Carboxy groups are often protectable as readily cleavable esters, such as the t-butyl or benzyl ester.
Examples of O-protected hydroxylamines for use in method (a) above include O-benzylhydroxylamine, O-4-methoxybenzylhydroxylamine, O-trimethylsilylhydroxylamine, and O-tert-butoxycarbonylhydroxylamine.
Examples of O,N-diprotected hydroxylamines for use in method (a) above include N,O-bis(benzyl)hydroxylamine, N,O-bis(4-methoxybenzyl)hydroxylamine, N-tert-butoxycarbonyl-O-tert-butyldimethylsilylhydroxylamine, N-tert-butoxycarbonyl-O-tetrahydropyranylhydroxylamine, and N,O-bis(tert-butoxycarbonyl)hydroxylamine.
Compounds of formula (II) may be prepared by a process comprising: coupling an acid of formula (III) or an activated derivative thereof with an amine of formula (IV) 
wherein R, R1, R2, R3, and R4 are as defined in general formula (I) except that any substituents in R1, R2, R3, and R4 which are potentially reactive in the coupling reaction may themselves be protected from such reaction, and R11 represents a hydroxy protecting group, and subsequently removing the protecting group R11 and any protecting groups from R1, R2, R3, and R4.
Compounds of the invention wherein R4 is a thioester may be prepared by coupling a compound of formula (IIIA) or an activated derivative thereof 
wherein R1 and R2 are as defined in general formula (I) and R12 and R13 are respectively N- and O-protecting groups, with a compound of formula (IV) above wherein R4 is a thioester group, and selectively removing the O- and N-protecting groups from the hydroxamic acid group.
Active derivatives of acids (III) and (IIIA) include activated esters such as the pentafluorophenyl ester, acid anhydrides and acid halides, chlorides. Suitable hydroxy protecting groups may be selected from those known in the art.
Amino acid esters and thioesters of formula (IV) are either known or are prepared by routine known synthetic methods.
As mentioned above, compounds of formula (I) above, and those of formula (I) excluded by the provisos in the definition of formula (I) above, are useful in human or veterinary medicine since they are active as inhibitors of the proliferation of cancer cells. The utility of the invention therefore lies in the treatment of cancers, such as those caused by over-proliferation of lymphoma, leukemia, myeloma, adenocarcinoma, carcinoma, mesothelioma, teratocarcinoma, choriocarcinoma, small cell carcinoma, large cell carcinoma, melanoma, retinoblastoma, fibrosarcoma, leiomyosarcoma, glioblastoma or endothelioma cells. It will be understood that different compounds (I) will have differing potencies as proliferation inhibitors depending on the type of cancer being treated.
The activity of any particular compound (I) in inhibiting proliferation of any particular cell type may be routinely determined by standard methods, for example analogous to those described in the Biological Example herein. From the fact that compounds (I) which are poorly active as inhibitors of MMPs are nonethless active in inhibiting proliferation of cancer cells, it is inferred that their utility in treating cancers is different from or supplementary to the utility of effective MMP inhibitors in the treatment of cancers.
Another aspect of the invention provides a compound selected from the group consisting of:
2(R or S)-[2-R-(S-Hydroxy-hydroxycarbamoyl-methyl)-4-methyl-pentanoylamine]-2-phenyl-ethanoic acid cyclopentyl ester,
2(R or S)-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-2-phenylethanoic acid isopropyl ester,
2(R or S)-[2R-(S-Hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3-phenylethanoic acid cyclopentyl ester,
2(R or S)-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-2-(4-methoxyphenyl)ethanoic acid cyclopentyl ester,
2(R or S)-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-2-(thien-2-yl)ethanoic acid cyclopentyl ester,
2(R or S)-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-2-(thien-3-yl)ethanoic acid cyclopentyl ester, and a pharmaceutically or veterinarily acceptable salts, hydrates or solvates thereof.
The 2-S diastereomers of the above compounds are especially preferred.
It is understood that different compounds of the invention will have differing potencies as proliferation inhibitors depending on the type of cancer being treated. The activity of any particular compound of the invention in inhibiting proliferation of any particular cell type may be routinely determined by standard methods, for example analogous to those described in the Biological Example herein.
In a further aspect of the invention there is provided a pharmaceutical or veterinary composition comprising a compound of the invention as defined by reference to formula (IB) above, together with a pharmaceutically or veterinarily acceptable excipient or carrier. One or more compounds of the invention may be present in the composition together with one or more excipient or carrier.
The compounds with which the invention is concerned may be prepared for administration by any route consistent with their pharmacokinetic properties.
Orally administratable compositions may be in the form of tablets, capsules, powders, granules, lozenges, and liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions. Tablets an capsules for oral administration may be in unit dose presetnation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants for example potato starch, or acceptable wetting agents such as sodium laurylsulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice.
Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.
For topical application to the skin, the drug may be made up into a cream, lotion or ointment. Cream or ointment formulations, which may be used for the drug, are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceuticals such as the British Pharmacopoeia.
The active ingredient may also be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the drug can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.
It will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
Although the present invention and that of International Application PCT/GB97/02398 are not limited by any particular theory of mechanism of action, the anti-proliferative effect is presently believed to be due to the inherent ability of the compounds, which inhibit one or more enzymes residing within the proliferating cells. According to this theory of the mechanism of activity, it follows that the most active compounds are those which are inherently most capable of passing through the cell wall into the cell interior. Since the compounds are esters (or thioesters) it is believed that esterases within the cell wall hydrolyze the compounds to the corresponding acids, and that the anti-proliferative activity may ultimately be due to inhibition of one or more intracellular enzymes by the unhydrolyzed ester, by the corresponding acid, or by a combination of both. If this is correct, the later inherent property is the more likely, since it is known that the rate of enzymatic hydrolysis may be more or less rapid, depending on the particular ester and the esterases present in the microenvironment of the ester within the cell. Cell penetrating pro-drugs of the acids or cell penetrating formulations of the acids are therefore expected to have the anti-proliferative effects of the esters described herein and in PCT/GB97/02398. Thus, while known MMP inhibitors work from without the target cell, the esters and thioester compounds of the present invention work from within the target cell.
The following examples 1-3 and 5-43 illustrate various embodiments of the invention. Example 4 describes the preparation of a compound for comparison with those of the invention. The following abbreviations have been used in the examples
DCFxe2x80x94Dichloromethane
DMFxe2x80x94N,N-Dimethylformamide
NMMxe2x80x94N-Methylmorpholine
TFAxe2x80x94Trifluoroacetic acid
HOBTxe2x80x941-Hydroxybenzotriazole
MeOdxe2x80x94methanol-d4 (CD3OD)
Column chromatography was performed with flash grade silica gel. 1H-NMR and 13C-NMR were recorded on a Bruker AC 250E spectrometer at 250.1 and 62.9 MHz respectively. CDCl3methanol-d4 and dimethylsulphoxide-d6. (DMSO-d6) were used as solvents and internal reference and spectra are reported as xcex4 ppm from TMS.